Throughout this specification, various references are identified by a number in parentheses. The citation to the reference corresponding to the identified number can be found in the section entitled References Cited preceding the claims. The references in that section are hereby incorporated by reference.
Esterases (also referred to as lipases) are enzymes that cleave triglycerides (fats or lipids) or esters into carboxylic acids (fatty acids) and mono- and di-glycerides. For an explanation of the slightly different definitions given to lipases and esterases see Siezen, R. J. and van den Berg, (37). A pregastric esterase is an esterolytic or lipolytic enzyme secreted by the oral tissues of mammals. Animal esterases in an unpurified form called rennet have been used in the production of dairy food products and, in particular, the production of enzyme modified cheeses or EMCs. (8), (9), (10), (17), (18), (33), (40), and (41). In particular, cheeses like Romano and Provolone have a xe2x80x9cpepperyxe2x80x9d or xe2x80x9cpiccantexe2x80x9d flavor due to the fatty acid composition created by the enzyme in the rennet paste. (26), (37).
Traditionally EMCs are prepared by esterases obtained from the gullet of slaughtered animals from which a rennet paste or powder is obtained. The rennet is used to treat whey to impart flavor into the cheese product. Kid pregastric estersase (kPGE or kid PGE) in rennet paste is contaminated with proteins which are found in the gullet of the kid and other substances used in the preparation of the rennet. It would be useful to have an uncontaminated kPGE to produce EMC""s. Such EMC""s could be produced in a manner acceptable to kosher and vegetarian consumers. A recombinant kPGE (rKPGE) could be produced in very pure form free of the other substances found in the present commercial rennet formulations.
The present invention provides kPGE and derivative polypeptides which are capable of being produced by genetic recombination and used to produce EMCS. This invention further provides nucleic acid sequences encoding kPGE and derivative polypeptides which can be used to create recombinant host cells that express kPGE and derivative polypeptides. A further subject of the present of invention is a fusion polypeptide called polyHis-enterokinase which increases expression of esterases and lipases when fused to the N-terminal of the esterase or lipase. This invention also provides a method for treating animals with an esterase or lipase deficiency by administering rkPGE to the animal in a therapeutically effective amount.